Sebastian Barg

Ongoing projects:

Molecular architecture of the insulin granule release site

Every ß-cell contains thousands of secretory granules that store insulin. When blood glucose is elevated, these granules undergo regulated exocytosis and release the hormone into the blood stream. Before this can happen, granules have to reach the plasma membrane, where they “dock” and then assemble the exocytosis machinery. When insulin is released, these steps quickly become limiting for how much insulin is released.

The docking process is not understood in molecular terms, but many of the proteins involved have been identified. One hypothesis that we are currently testing is that some of these proteins (including t-SNAREs) pre-assemble at small hotspots in the plasma membrane. These hotspots, perhaps related to lipid rafts, may then recruit granules and act as “launching pads” for exocytosis. How do cells compartmentalize their plasma membrane to organize such sites? Which proteins are recruited to these hotspots, when, and at how many copies? And how are docking sites regulated and what distinguishes release-ready granules from those that are merely docked?

Fig 1. Quantification of syntaxin and munc18 during granule docking. (A-B) A single granule (gr)is docking at the plasma membrane. At the same time syntaxin-EGFP (syx) and munc18-EGFP (m18) accumulate at the docking site. (C) Successful docking: The granule approaches the plasma membrane where it induces clustering of free syntaxin and munc18 molecules. Clustered syntaxin then stabilizes the docked state. Later, the site matures by recruiting additional proteins, allowing exocytosis. (D) Transition to the docked state does not occur when syntaxin/munc18 cluster formation fails.

Fig 2. Single molecule imaging in live cells. (A-B) Schematic of the experiment; individual SNARE proteins and secretory granules are labeled using fluorescent proteins, and imaged with a custom-built two color TIRF microscope. Excitation is limited to the narrow space containing docked granules and the plasma membrane (C) Image sequence showing a single syntaxin1A-EGFP molecule (green) near a secretory granule (red), recorded at 50Hz; every 5th frame is shown. (D) trajectories of single syntaxin molecules. Note that the molecules represented by colored tracks pause their motion near docked granules (yellow circles), as if captured.

For further information about this research group please contact Sebastian Barg